Methods of enhancing antigen-specific T cell responses

ABSTRACT

Recombinant polynucleotides are provided that confer at least partial immunity on an individual to an infectious intracellular pathogenic agent. The recombinant polynucleotides encode a costimulatory factor and/or a target antigen polypeptide. The immune response that confers the immunity results from the expression of both polypeptides in an antigen presenting cell in the individual. The immunity is to the pathogenic agent that naturally encodes the target antigen polypeptide.

CROSS-REFERENCE TO RELATE APPLICATIONS

The present application is a national phase application under 35 U.S.C.§371 of PCT/US94/04367, filed Apr. 20, 1994, which is acontinuation-in-part application of U.S. Ser. No. 08/049,259, filed Apr.20, 1993, now abandoned, the contents of which are incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to methods of preventing new infections, curingcurrent infections and treating chronic infections caused byintracellular infectious agents. The methods encompass using genetherapy to enhance the interaction between ligands that costimulate Tcell activation. More specifically, this invention relates to materialsand methods of gene therapy in which genes encoding an antigen from aninfectious agent and ligands that costimulate T cell activation areco-expressed in the cells of the host animal to prevent or treat chronicinfection. This method stimulates or enhances immune responses Toantigens of infectious agents by enhancing the antigen-specific T cellresponse thus providing protection against new infection or terminatingchronic infection.

BACKGROUND OF THE INVENTION

Many infectious agents are capable of causing infection and disease in ahost organism; examples of such infectious agents include protozoa,fungi, bacteria, viruses and even worms. Of these infectious agents,those with the mechanism to act intracellularly pose the mostsignificant challenge for prevention and treatment. This is particularlytrue where the host of the intracellular infectious agent is a mammal,such as man.

Mammals have a complex immune system which has several differentmechanisms for defending against an invasion by an infectious agent.Under normal circumstances, the immune system functions to ultimatelyeliminate the infectious agent from the mammalian host's system. Mostinfections are self-limiting because the mammalian immune system isgenerally able to eliminate the infectious agents. Problems arise,however, when the immune system is unable to, or does not, respond tothe presence of an infectious agent. In this scenario, infections canbecome chronic and can persist for many years or even the life-time ofthe infected human or animal, often resulting in serious disease.

Traditional, standard vaccines expose the immune system to a foreignantigen such as those of infectious agents to elicit an antigen-specificimmune response. The immune response is most often humoral and notcellular, that is, it results in production of antibodies but not Tcell-based immunity as discussed below. Effective vaccination preventsinfection or modifies diseases resulting from infection caused by theinfectious agent to which the vaccine is directed.

Immune responses that elicit neutralizing antibodies are sufficient forpreventing infections with certain viruses. Murphy et al. (1990). Thus,infection by these viruses can be prevented by vaccination with antigensthat elicit neutralizing antibodies. For some viruses and non-virusinfectious agents, however, neutralizing antibodies either cannot beelicited or, if elicited, are insufficient to prevent infection and/ordisease progression.

Two traditional vaccine types are killed viruses and live attenuatedviruses. Killed viruses consist of either whole virions or otherinfectious agents the infectivity of which has been inactivated. Thesevaccines may also consist of antigenic subunits or component parts ofvirions or other infectious agents. These vaccines are administered byparenteral inoculation or exposure to mucous membranes. Live attenuatedvaccines consist of live virus or other infectious agents withgenetically altered virulence. When used for vaccination, these vaccinescause a reduced form of the disease, or no disease at all.

A third vaccine approach has been investigated and used experimentallybut is not yet in clinical use. This approach is the utilization of liveagents such as viruses or bacteria as vectors to express vaccineantigens of heterologous infectious agents. A gene or nucleotidesequence encoding the antigen is expressed by the vector and when usedin vaccination, exposes the host to the antigen. An immune response tothe antigen is expected to protect the host from the infectious agentfrom which the antigen was derived.

Examples of killed vaccines include tetanus toxoid, influenza virussubunit, rabies, polio and HBV. Examples of live attenuated vaccinesinclude Salmonella typhi Ty 21-a and live vaccines for polio, measles,rubella, mumps, smallpox and yellow fever viruses. Examples of livevectors used to express heterologous vaccine antigens include vacciniavirus, adenovirus, adeno-associated virus and S. typhi Ty 21-a strain.

Examples of chronic infections associated with significant morbidity andearly death include the two human hepatitis viruses, hepatitis B virus(HBV) and hepatitis C virus (HCV) which cause chronic hepatitis,cirrhosis and liver cancer. HBV infection in man closely parallels theinfections caused by the closely related hepadnaviruses in certainanimals including ground squirrel hepatitis virus (GSHV) which infectsthe Beechey ground squirrels, woodchuck hepatitis virus (WHV) whichinfects woodchucks, and duck hepatitis B virus (DHBV) which infectsducks. Robinson, in, Virology, 2nd Ed., ed. B. Fields, Raven Press, NewYork, pp. 2137-2169 (1990).

Additional examples of chronic infections-in man caused by viralinfectious agents include those caused by the human retroviruses: humanimmunodeficiency viruses (HIV-1 and HIV-2), which cause acquired immunedeficiency syndrome (AIDS); and human T lymphotropic viruses (HTLV-1 andHTLV-2) which cause T cell leukemia and myelopathies. Many otherinfections such as human herpes viruses including the herpes simplexvirus (HSV) types 1 and 2, Epstein Barr virus (EBV), cytomegalovirus(CMV), varicella-zoster virus (VZV) and human herpes virus 6 (HHV-6) arenot eradicated by host mechanisms, but rather become chronic and in thisstate may cause disease. Chronic infection with human papilloma virusesis associated with cervical carcinoma. Numerous other viruses and otherinfectious agents replicate intracellularly and may become chronic whenhost defense mechanisms fail to eliminate them. These include pathogenicprotozoa (e.g., Pneumocystis carinii, Trypanosoma, Leishmania, Malariaand Toxoplasma gondii), bacteria (e.g., mycobacteria, salmonella andlisteria), and fungi (e.g., candida and aspergillus).

Treatment of chronic virus infections resulting in significant clinicalbenefit has not been successful largely because such treatments fail toterminate the infection or eliminate the virus. Previous treatmentsinclude administration of small chemical compounds such as nucleosideanalogs or biologically active proteins such as interferons. Thesetreatments inhibit virus replication but do not eliminate virus fromcells or virus infected cells themselves and may result in limiteddisease improvement only during their administration. Unfortunately,viral infections are often exacerbated when administration of the drugis discontinued.

Common antiviral treatments include nucleoside analogues such asazidothymidine (AZT), dideoxyinosine (DDI) and dideoxycytodine (DDC) forchronic HIV infection and associated AIDS, and adenine arabinoside(araA) for HBV infection and associated liver disease. Interferonssimilarly suppress HBV and HCV during therapy of chronic infection butthe virus usually returns to pretreatment levels and the disease isexacerbated when therapy is discontinued.

One important mechanism employed by the mammalian immune system forcontrolling and ultimately eliminating ongoing infections byintracellular infectious agents is the activated cellular immuneresponse by activation of certain T cells. T cell activation results ina cytotoxic T lymphocyte (CTL) response directed at viral (or otherintracellular infectious agents) antigens on the infected cell surfaceand elimination of the infected cells. Guilhot et al., J. Virol.,66:2670-2678 (1992). This ultimately results in the elimination ofinfected cells and the infectious agent within the infected cells. For ageneral discussion of immune responses to viral infectious agents, seeWhitton et al., in, Virology, 2nd Ed., ed. B. Fields, Raven Press, NewYork, pp. 369-381 (1990); and Roitt, Essential Immunology, 7th Ed.(1991).

Activation of T cells occurs when the T cell receptor (TCR) forms aternary complex with an antigen peptide complexed with a self-MHC (majorhistocompatibility complex) molecule on the surface of professionalantigen-presenting cells (APC). Professional APCs include macrophagesand activated B cells. Townsend et al., Cell, 44:959-968 (1986);Townsend et al., Ann. Rev. Immunol., 7:601-624 (1989); Bjorkman et al.,Nature, 329:512-518 (1987); and Jardetsky et al., Nature, 353:326-329(1991). The ternary complex allows for a costimulatory signal to passbetween the cells. Activation of T cells requires not only recognitionof the antigen peptide-MHC complex by the TCR, but also the interactionof "costimulatory factors," located on the surface 6f APCs, withspecific molecules on the surface of the T cells, the "costimulatoryligand." Freeman et al., J. Exp. Med., 174:625-691 (1991). As usedherein, costimulatory factors on APCs include, but are not limited to,the B7-1 protein which specifically binds CD28 and CTL-4 proteins on thesurface of T cells and the B7-2 and B7-3 proteins which bind CTLA-4 in Tcell activation. Boussiotis et al., Proc. Natl. Acad. Sci. USA,90:11059-11063 (1993). B7 ligands are expressed exclusively byprofessional APCs. Freeman et al., J. Exp. Med., 174:625-631 (1991);Razi-Wolf et al. (1992); and Larsen et al. (1992). The interaction ofCD28 and B7-1 has been shown to be essential for T cell activation.Jenkins et al., J. Immunol., 140:3324-3330 (1988); Linsley et al.(1990); Linsley et al., J. Exp. Med., 173:721-730 (1991); Gimmi et al.,Proc. Natl. Acad. Sci. USA, 88:6575-6579 (1991); Jenkins et al., J.Immunol., 147:2461-2466 (1991); and Harding et al. (1992).

One of the costimulatory molecules found on the APC is the B7 proteinwhich is the ligand for the T cell surface differentiation antigen CD28.B7 expression, typically effected by professional APCs, has been foundto be of critical importance to the activation of naive T cells. Larsenet al., J. Exp. Med., 176:1215-1220 (1992). Other studies have shownthat there is a direct relationship between the increase in functionalactivity of the T cell with the increase in B7 expression. Razi-Wolf etal., Proc. Natl. Acad. Sci. USA, 89:4210-4214 (1992). T cells arerendered anergic when they, encounter antigen peptides on cells lackingthe costimulatory ligand for which CD28 is a receptor. Harding et al.,Nature, 356:607-609 (1992).

Viral antigens can be degraded in infected cells when specific viralantigen peptide fragments that bind MHC class I molecules are presentedat the cell surface where they serve as targets for MHC restricted CTL.Whitton et al. (1990). However, most infected cells in virally infectedhosts are not professional APCs expressing costimulatory proteins.Furthermore, infected cells lacking costimulatory molecules such asB7-1, B7-2, B7-3 may not elicit an effective cellular immune response.When such mechanisms are inadequate and fail to eliminate the agent,infections may persist and become chronic.

Some immunologic mechanisms, such as CTL responses, that are involved inresolving an ongoing infection caused by an intracellular infectiousagent, may also play a role in preventing new infections. While otherimmunologic mechanisms, such as a neutralizing antibody, clearly appearto be more important for preventing certain viral infections than forresolving an ongoing infection. In the case of neutralizing antibodies,antibodies directed against surface antigens can act to neutralize theinfectivity of the virus by promoting viral aggregation and ultimatelyremoval of the virus from the bloodstream. Viral infections that aretypically blocked by a virus neutralizing antibody, can also beprevented by immunization with antigens that elicit the sameneutralizing antibodies. However, for other intracellular infectiousagents, a neutralizing antibody cannot be elicited or, when it iselicited, is insufficient for protection. Nonetheless, protection can beconferred by the introduction of an appropriate immunizing agent. Insuch cases, the cellular immune mechanisms appear to facilitate abilityof the immunizing agent to protect against infection. In order for theimmune response to result in protection, the immunizing agent mustelicit a strong and persistent CTL response. Murphy et al., in,Virology, 2nd Ed., ed. B. Fields, Raven Press, New York, pp. 469-502(1990).

Thus, there has been a need for a method of enhancing the immuneresponse to intracellular infectious agents, such as viruses, byeliciting a strong CTL response. The present invention satisfies thatneed.

SUMMARY OF THE INVENTION

The present invention provides materials and methods for enhancing theimmune response of an individual to an intracellular infectious agent,such as a virus, protozoan, fungus, or bacteria. The materials includevectors encoding a costimulatory factor for T cell activation(hereinafter "costimulatory factor") and a "target antigen" from theinfectious agent. The vector, either as a polynucleotide or packagedinto a virus assembly, is either directly administered to the individualto be treated, or alternatively, is inserted into a cell which is thenadministered to the individual to be treated. The administration of thegenetic information encoding the costimulatory factor and the targetantigen enhances the immune response by, inter alia, eliciting thegeneration of CTLs directed at cells infected with the infectious agentfrom which the target antigen is derived.

Accordingly, one embodiment of the invention is a recombinantpolynucleotide comprised of a region encoding a costimulatory factoroperably linked to a transcriptional control region and furthercomprised of a region encoding a target antigen polypeptide operablylinked to a transcriptional control region. Expression of thecostimulatory factor and target antigen polypeptide in an individualconfers at least partial immunity in the individual to an intracellularinfectious agent that naturally encodes the target antigen polypeptide.The recombinant polynucleotide my be comprised of a viral vector orother vectors as described below.

Another embodiment of the invention is a host cell transformed with arecombinant polynucleotide comprised of a region encoding acostimulatory factor operably linked to a transcriptional control regionand with a recombinant polynucleotide comprised of a region encoding atarget antigen polypeptide operably linked to a transcriptional controlregion. Expression of the costimulatory factor and target antigenpolypeptide in the individual confers at least partial immunity to anintracellular infectious agent that naturally encodes the target antigenpolypeptide.

Still another embodiment of the invention is a method of using arecombinant polynucleotide comprised of a region encoding acostimulatory factor operably linked to a transcriptional control regionand with a recombinant polynucleotide comprised of a region encoding atarget antigen polypeptide operably linked to a transcriptional controlregion, the method comprising administering the recombinantpolynucleotide to an individual in a therapeutically effective amount;and determining a lessening of a physical symptom associated with aresponse to infection by an intracellular pathogen that naturallyencodes the target antigen.

Yet another embodiment of the invention is a method of using theabove-described recombinant polynucleotides comprising transforming ahost cell with the polynucleotide. Also claimed are host cells preparedby this method, and progeny thereof.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a line drawing of two virus vectors.

DETAILED DESCRIPTION OF THE DRAWING

The critical role of costimulatory factors is to make cells expressingthe desired target antigens, such as vital antigens, effective APCs inorder to facilitate T cell activation. By creating a system whichexpresses a costimulatory factor (or a functional portion thereof)concurrently with a target antigen, the resulting response leads toelimination of infected host cells and stronger protection of uninfectedhosts cells against new infections.

The invention teaches that immunotherapy that introduces into anindividual genetic information encoding the functional portions of acostimulatory factor and a target antigen can be used to activate and/orenhance the immune response of an infected mammalian host. Theenhancement includes a cellular response to infections caused by theintracellular infectious agent from which the target antigen is derived,and an induced protective immune response in uninfected cells to preventnew infection to the infectious agent.

The following terms used herein are defined as follows.

The term "polypeptide" refers to a polymer of amino acids and does notrefer to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),polypeptides with substituted linkages, as well as other modificationsknown in the art, both naturally occurring and non-naturally occurring.

"Transformation", as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for the insertion, for example, direct uptake, transduction,f-mating or electroporation. The exogenous polynucleotide may bemaintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host cell genome.

"Treatment" as used herein refers to prophylaxis and/or therapy. Theeffectiveness of a treatment can be determined by the alleviation of oneor more symptom normally associated with a disease caused by anintracellular pathogen.

"Intracellular pathogen" refers to an agent capable of causing a diseasestate in a susceptible individual, in which part or all of thereplicative cycle of the pathogen occurs within the cells of an infectedindividual. Intracellular pathogens include, for example, protozoa,fungi, bacteria, and viruses.

"Helper cells" or "T_(H) cells" are a functional subclass of T cellswhich can help to generate cytotoxic T cells and cooperate with B cellsin the production of an antibody response. Helper cells Usuallyrecognize antigen in association with class II MHC molecules.

An "antigen specific T cell clone" is comprised of the progeny of asingle cell; the cells in this type of clone are of the same phenotypeand are all targeted towards the same antigen. Methods of preparingantigen-specific T cell clones are known in the art.

The term "recombinant expression vector" refers to a replicable unit ofDNA or RNA in a form capable of being introduced into a target cell bytransformation, electroporation, transduction or Viral infection, andwhich codes for the expression of a heterologous structural codingsequence, for example, a cytokine, which is transcribed into mRNA andtranslated into protein under the control of elements having aregulatory role in gene expression. Such vectors will preferably alsocontain appropriate transcription and translation control sequences,including initiation sequences operably linked to the coding sequence.

"Recombinant," as used herein, means that a particular DNA sequence isthe product of various combinations of cloning, restriction, andligation steps resulting in a construct having a structural codingsequence distinguishable from homologous sequences found in naturalsystems. Generally, DNA sequences encoding the structural codingsequence, for example cytokines, can be assembled from cDNA fragmentsand short oligonucleotide linkers, or from a series of oligonucleotides,to provide a synthetic gene which is capable of being expressed in arecombinant transcriptional unit. Such sequences are preferably providedin the form of an open reading frame uninterrupted by internalnontranslated sequences, or introns, which are typically present ineukaryotic genes. Genomic DNA containing the relevant sequences couldalso be used. Sequences of non-translated DNA may be present. 5' or 3'from the open reading frame, where such sequences do not interfere withmanipulation or expression of the coding regions.

"Recombinant host cells", "host cells", "cells", "cell lines", "cellcultures", and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be, or have been, used as recipients for recombinant vectorsor other transfer polynucleotides, and include the progeny of theoriginal cell which has been transfected. It is understood that theprogeny of a single cell may not necessarily be completely identical inmorphology or in genomic or total DNA Complement as the original parent,due to natural, accidental, or deliberate mutation.

A CTL is "cytolytically specific for" cells expressing antigens if theCTL is capable of selectively recognizing and lysing the cells bearingthe antigen. A CTL is "cytolytically reactive against" cells expressingantigens if the CTL is capable of lysing the cells bearing the antigen,without regard to its ability to selectively recognize such cells.

"Antigen specific expression" refers to expression that occurs when theT cell recognizes its cognate antigen.

"Cognate antigen" refers to an antigen, a peptide of which whenassociated with an MHC molecule forms a ligand that binds to alymphocyte that recognizes it and causes triggering of signals for theeffector function of the cell and/or for proliferation.

"Target antigen" refers to an antigen that when expressed in a cell canelicit a CTL response directed at cells expressing that antigen.

An "agretope" is the portion of an antigen or antigenic fragment whichallows it to bind to an MHC molecule.

An "activated lymphocyte" is one that as a result of binding of acognate antigen peptide:MHC molecule is producing polypeptidestimulatory factors (including, for example, cytokines) at a level thatis elevated relative to the lymphocyte without the bound ligand.

A "transcriptional regulatory region" encompasses all the elementsnecessary for transcription, and may include elements necessary forregulation and cell-specific transcription. Thus, a transcriptionalregulatory region includes at least the promoter sequence, and my alsoinclude other regulatory sequences such as enhancers, and transcriptionfactor binding sites.

"Operably linked" refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. For instance, a promoter is operably linked to a codingsequence if the promoter affects its transcription or expression.

The term "recombinant" polynucleotide or nucleic acid refers to onewhich is not naturally occurring, or is made by the artificialcombination of two otherwise separated segments of sequence. Thisartificial combination is often accomplished by either chemicalsynthesis means, or by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques. Such isusually done to replace a codon with a redundant codon encoding the sameor a conservative amino acid, while typically introducing or removing asequence recognition site. Alternatively, it is performed to jointogether nucleic acid segments of desired functions to generate adesired combination of functions.

"Regulatory Sequences" refer to those sequences normally associated with(for example within 50 kb) of the coding region of a locus which affectthe expression of the gene (including transcription of the gene, andtranslation, splicing, stability, or the like of the messenger RNA).Regulatory sequences include, inter alia, promoters, enhancers, splicesites and polyadenylation sites.

The "sense strand" of a nuclei acid contains the sequence that hassequence homology to that of the cognate mRNA. The "anti-sense strand"contains a sequence which is complementary to that of the "sensestrand".

An "individual" as used herein refers to vertebrates, particularlymembers of the mammalian species, and includes, but is not limited to,domestic animals, sports animals, and primates, including humans.

"Immunization" refers conferring a state of immunity upon an individualby administration of a therapeutic or prophylactic agent.

By "immunity" is meant a lessening and/or prevention of one or morephysical symptoms associated with a response to infection by thepathogen from which the target antigen was derived.

An "immune response" to a composition or vaccine is the development inthe host of a cellular and/or antibody-mediated immune response to theintracellular infectious agent that encodes the target antigen. Usually,such a response comprises the individual producing CTLs and/or B cellsand/or a variety of classes of T cells directed specifically to APCsexpressing the target antigen.

A "therapeutically effective amount" of a composition is a dosesufficient to induce an immune response and/or to confer immunityagainst an intracellular infectious agent that naturally encodes thetarget antigen.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, Fritsch, and Maniatis; MOLECULAR CLONING: A LABORATORY MANUAL,Second Edition (1989), OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait Ed., 1984),ANIMAL CELL CULTURE (R. I. Freshney, Ed., 1987), the series METHODS INENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIANCELLS (J. M. Miller and M. P. Calos Eds. 1987), HANDBOOK OF EXPERIMENTALIMMUNOLOGY, (D. M. Weir and C. C. Blackwell, Eds.); CURRENT PROTOCOLS INMOLECULAR BIOLOGY, (F. M. Ausubel, R. Brent, R. E. Kingston, D. D.Moore, J. G. Siedman, J. A. Smith, and K. Struhl Eds. 1987); and CURRENTPROTOCOLS IN IMMUNOLOGY (J. E. Coligan, A. M. Kruisbeek, D. H.Marguiles, E. M. Shevach and W. Strober Eds. 1991). All patents, patentapplications, and publications mentioned herein, both supra and infra,are hereby incorporated herein by reference.

In accordance with the invention, APCs which express a target antigenand are capable of stimulating a T cell response, preferably a CTLresponse, are created either in vivo or in vitro by the insertion of oneor more recombinant polynucleotides containing a sequence encoding atleast one costimulatory factor and at least one target antigenpolypeptide, such that the costimulatory factor(s) and the targetantigen polypeptide(s) are expressed within the recipient host cell.

The costimulatory factor expressed from the recombinant polynucleotidewill include at least a portion of the protein sufficient to allowbinding of the cell expressing the costimulatory factor to itscostimulatory ligand. Methods for determining such binding are known inthe art. See, for example, Linsley et al. who describe a procedurewherein the cells to be tested are labeled with ⁵¹ Cr and then incubatedwith either CD28⁺ and CD28³¹ CHO cells, and the adhesion of the labelledcells to the CHO cells is determined by ⁵¹ Cr release. Proc. Natl. Acad.Sci. USA, 87:5031-5035 (1990). The extracellular domains andtransmembrane domains of the costimulatory factors are usually includedin the polypeptide, and in preferred embodiments, the entirecostimulatory factor is encoded. The sequences for polynucleotidesencoding costimulatory factors and the functional domains are known, andare described in, for example, Linsley et al. (1990) (human) and Freemanet al., J. Immunol., 143:2714-2722 (1989) (mouse).

The target antigen polypeptide expressed from the recombinantpolynucleotide is all or a fragment of a target antigen that isnaturally encoded in the pathogenic intracellular microorganism againstwhich an enhanced or augmented immune response is desired, and iscomprised of one or more agretopes from that microorganism. Targetantigens are preferably from viruses, and particularly viruses thatresult in chronic infections, for example, the hepadnaviruses (includingHBV), the lentiviruses (including HIV), herpesviruses (including HSV-1,HSV-2, EBV, CMV, VZV, and HHV-6), and the flaviviruses/pestiviruses(including HCV). Also included as viruses that cause chronic viralinfections are human retroviruses, for example, human T lymphotropicviruses (HTLV-1 and HTLV-2) that cause T cell leukemia and myelopathies.Other organisms that cause chronic infections include, for example,pathogenic protozoa, (e.g., Pneumocystis carinii, trypanosoma, malariaand Toxoplasma gondii), bacteria (e.g., mycobacteria, salmonella andlisteria) and fungi (e.g. candida and aspergillus).

The nucleotide sequences of a number of these viruses, includingdifferent species, strains, and isolates are known in the art. Forreviews see: Robinson (1990) (Hepadnaviridae); Levy, MicrobiologicalReviews, 57:183-289 (1993) (HIV); and Choo et al., Seminars in LiverDisease, 12:279-288 (1992) (HCV). Particularly suitable target antigensare those which induce a T cell response, and particularly aCTL-response during infection. These may include, for example, from HBV,the core antigen, the E antigen, and the surface antigen (HBsAg).Polynucleotide sequences for HBsAg including the pre-S1, pre-S2 and Sregions from a variety of surface antigen subtypes are known in the art.See, for example, Okamoto et al., J. Gen. Virol., 67:1383-1389 (1986);Genbank accession numbers D00329 and D00330. The polynucleotidesequences encoding HIV glycoprotein gp160 and other antigenic HIVregions are known in the art. Lautenberger et al., Nature, 313:277-284(1985); Starcich et al., Cell, 45:637-648 (1986); Wiley et al., Proc.Natl. Acad. Sci. USA, 83:5038-5042 (1986); and Modrow et al., J. Virol.,61:570-578 (1987).

It is within the scope of the invention to include nucleotides encodingtwo or more target antigen polypeptides that may or may not be fused.The two target antigen polypeptides may be from the same pathogenicintracellular microorganism, or when it is desirable to enhance theimmune response to more than one microorganism, from differingmicroorganisms.

The sequences encoding the costimulatory factor and the target antigenpolypeptide are operably linked to a transcriptional control region.Transcriptional control regions are known in the art, and include, forexample, regions isolated from the following: the human cytomegalovirus(HCMV) IE94 promoter region (Boshart et al., Cell, 41:521-530 (1985));the human IL-2 gene (Fujita et al., Cell, 46:401-407 (1986)); the humanIFN-γ gene (Ciccarone et al., J. Imunol., 144:725-730 (1990)); the humanIL-3 gene (Shoemaker et al., Proc. Natl. Acad. Sci. USA, 87:9650-9654(1990)); th& human IL-4 gene (Arai et al., J. Immunol., 142:274-282(1989)); the human lymphotoxin gene (Nedwin et al., Nucl. Acids. Res.,13:6361-6373 (1985)); the human granulocyte-macrophage CSF (GM-CSF) gene(Miyatake et al., EMBO J., 4:2561-2568 (1985)); the human perforin gene(Lictenheld et al., J. Immunol., 143:4267-4274 (1989)); the human 519gene (Manning et al., J. Immunol., 148:4036-4042 (1992)); the humangranzyme B (CTLA-1) gene (Haddad et al., Gene, 87:265-271 (1990)); thehuman CTLA-4 gene (Harper et al., J. Immunol., 147:1397-1044 (1991));the human CGL-2 gene (Heusel et al., J. Biol. Chem., 266:6152-6158(1991)); the human granzyme H gene (Haddad et al., Int. Immunol.,3:57-66 (1990)); the human IL-2 receptor, α chain gene (Cross et al.,Cell, 49:47-56 (1987)); the Murine T cell activation 3 (TCA-3 ) gene(Wilson et al., J. Immunol., 141:1563-1570 (1988)); and the human CD69gene.

In some embodiments of the invention, the transcriptional controlregions are hybrids. For example, enhancer regions (e.g., from the HCMVIE transcriptional control region and/or from the SV40 early promoterregion) may be inserted upstream of the transcriptional control regions.Alternatively, or in addition, multimeric transcription factor bindingsites (e.g., NF-AT and/or NFKB) may be inserted into or upstream of thetranscriptional control regions, combining the upstream region of onewith the proximal region of the other. Secretion signals may also beincluded where appropriate, whether from a native protein or from othersecreted polypeptides of the same or related species, which allow theprotein to cross and/or lodge in cell membranes, and thus attain itsfunctional topology, or to be secreted from the cell.

In addition it is useful to include in the recombinant polynucleotides apositive marker that enables the selection of cells carrying therecombinant polynucleotide. The positive selectable-marker my be a genewhich, upon being introduced into the host cell expresses a dominantphenotype permitting positive selection of cells carrying the gene.Genes of this type are known in the art, and include, inter alia,hygromycin-B phosphotransferase gene (hph) which confers resistance tohygromycin B, the aminoglycoside phosphotransferase gene (neo or aph)from Tn5 which codes for resistance to the antibiotic G418, thedihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA),and the multi-drug resistance (MDR) gene.

The sequences encoding the costimulatory factor and the target antigenpolypeptide(s) may be on separate polynucleotides, but preferably are onthe same polynucleotide. These encoding sequences my also be under thecontrol of separate transcriptional control sequences, or under thecontrol of the same transcriptional control sequence.

In addition to transcriptional control regions, in some embodiments, thepolynucleotides encoding the costimulatory factor and target antigen(s)are in the form of recombinant expression vectors.

Techniques for nucleic acid manipulation are described generally, forexample, in Sambrook et al. (1989), Ausubel et al. (1987); and in AnnualReviews of Biochemistry, 61:131-156 (1992). Reagents useful in applyingsuch techniques, such as restriction enzymes and the like, are widelyknown in the art and commercially available from a number of vendors.

Large amounts of the polynucleotides used to create the cells of thepresent invention may be produced by replication in a suitable hostcell. The natural or synthetic polynucleotide fragments coding for adesired fragment may be incorporated into recombinant nucleic acidconstructs, typically polyucleotide constructs, Capable of introductioninto and replication in a prokaryotic or eukaryotic cell. Usually theconstructs will be suitable for replication in a unicellular host, suchas yeast or bacteria, but may also be intended for introduction to, withand without and integration within the genome, cultured mammalian orplant or other eukaryotic cell lines. The purification of nucleic acidsproduced by the methods of the present invention are described, e.g., inSambrook et al. (1989).

The polynucleotides used in the present invention may also be producedin part or in total by chemical synthesis, e.g., by the phosphoramiditemethod described by Beaucage and Carruthers, Tetra. Letts., 22:1859-1862(1981) or the triester method according to the method described byMatteucci et al., J. Am. Chem. Soc., 103:3185 (1981), and may beperformed on commercial automated oligonucleotide synthesizers. Adouble-stranded fragment may be obtained from the single strandedproduct of chemical synthesis either by synthesizing the complementarystrand and annealing the strand together under appropriate conditions orby adding the complementary strand using DNA polymerase with anappropriate primer sequence.

Polynucleotide constructs prepared for introduction into a prokaryoticor eukaryotic host cell for replication will typically comprise areplication system recognized by the host, including the intendedrecombinant polynucleotide fragment encoding the desired polypeptide.Such vectors may be prepared by means of standard recombinant techniqueswell known in the art and discussed, for example, in Sambrook et al.(1989) or Ausubel et al. (1987).

Preferably, during the cloning phase, the polynucleotide construct willcontain a selectable marker, a gene encoding a protein necessary for thesurvival or growth of a host cell transformed with the vector. Thepresence of this gene ensures the growth of only those host cells whichexpress the inserts. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxic substances, e.g.ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g. the gene encoding D-alanine racemase for Bacilli.The choice of the proper selectable marker will depend on the host cell,and appropriate markers for different hosts are well known in the art.

The recombinant polynucleotides encoding the costimulatory factor andtarget antigen polypeptide may be introduced into individuals in severalways. For example, the polynucleotides may be introduced ex vivo into ahost cell, for example, dendritic cells, or cells from a skin biopsy.The cells containing the recombinant polynucleotide may be used toconfer immunity to individuals. The cells are usually administered byinfusion, with each infusion in a range of at least 10⁶ to 10¹⁰cells/m², preferably in the range of at least 10⁷ to 10⁹ cells/m². Theclones may be administered by a single infusion, or by multipleinfusions over a range of time. However, since different individuals areexpected to vary in responsiveness, the type and amount of cellsinfused, as well as the number of infusions and the time range overwhich multiple infusions are given are determined by the attendingphysician or veterinarian, and can be determined by routine examination.

The polynucleotides encoding the costimulatory factor and the targetantigen polypeptide may be introduced into the desired cell ex vivo bymeans known in the art, including, for example/transformation,electroporation, lipofection, and transduction, including the use ofadeno-associated viral (AAV) vectors, and particularly using methods ofretroviral gene transfer known in the art.

Various infection techniques have been developed which utilizerecombinant infectious virus particles for gene delivery. Thisrepresents a preferred approach to the present invention. The viralvectors which have been used in this way include virus vectors derivedfrom simian virus 40 (SV40; Karlsson et al., Proc. Natl. Acad. Sci. USA,82:158 (1985)), adenoviruses (Karlsson et al., EMBO J., 5:2377 (1986)),AAV (Carter, Current Opinion in Biotechnology 1992, 3:533-539), andretroviruses (Coffin, in Weiss et al. (eds.), RNA Tumor Viruses, 2nded., Vol. 2, Cold Spring Harbor Laboratory, New York, 1985, pp. 17-71).Thus, gene transfer and expression methods are numerous but essentiallyfunction to introduce and express genetic material in mammalian cells.Several of the above techniques have been used to transducehematopoietic or lymphoid cells, including calcium phosphatetransfection (Berman et al. (1984)), protoplast fusion (Deans et al.(1984)), electroporation (Cann et al.., Oncogene, 3:123 (1988)), andinfection with recombinant adenovirus (Karlsson et al. (1986)); Reutheret al., Mol. Cell. Biol., 6:123 (1986); AAV (Carter (1985)) andretrovirus vectors (Overell et al., Oncogene 4:1425 (1989)).

Retroviral vectors provide a highly efficient method for gene transferinto eukaryotic cells which is the preferred method for the delivery ofthe polynucleotides of the invention. Moreover, retroviral integrationtakes place in a controlled fashion and results in the stableintegration of one or a few copies of the new genetic information percell.

Retroviruses are a class of viruses which replicate using avirus-encoded, RNA-directed DNA polymerase, or reverse transcriptase, toreplicate a viral RNA genome to provide a double-stranded DNAintermediate which is incorporated into chromosomal DNA of an arian ormammalian host cell. Most retroviral vectors are derived from murineretroviruses. Retroviruses adaptable for use in accordance with thepresent invention can, however, be derived from any avian or mammaliancell source. These retroviruses are preferably amphotropic, meaning thatthey are capable of infecting host cells of a broad host range ofseveral species, including humans.

A characteristic feature of retroviral genomes (and retroviral vectorsUsed as described herein) is the retroviral long terminal repeat, orLTR, which is an untranslated region of about 600 base pairs found inslightly variant forms at the 5' and 3' ends of the retroviral genome.When incorporated into DNA as a provirus, the retroviral LTR includes ashort direct repeat sequence at each end and signals for initiation oftranscription by RNA polymerase II and 3' cleavage and polyadenylationof RNA transcripts. The LTR contains all other cis-acting sequencesnecessary for viral replication.

A "provirus" refers to the DNA reverse transcript of a retrovirus whichis stably integrated into chromosomal DNA in a suitable host cell, or acloned copy thereof, or a cloned copy of unintegrated intermediate formsof retroviral DNA. Forward transcription of the provirus and assemblyinto infectious virus occurs in the presence of an appropriate helpervirus or in a cell line containing appropriate sequences enablingencapsidation without coincident production of a contaminating helpervirus. Mann et al., describe the development of "packaging" cell lines(e.g., ψ2) which can be used to produce helper-free stocks ofrecombinant retrovirus. Cell, 3:153 (1983).

Packaging cell lines contain integrated retroviral genomes which lacksequences required in cis for encapsidation, but which provide allnecessary gene product in trans to produce intact virions. The RNAtranscribed from the integrated mutant provirus cannot itself bepackaged, but these cells can encapsidate RNA transcribed from arecombinant retrovirus introduced into the same cell. The resultingvirus particles are infectious, but replication-defective, renderingthem useful vectors which are unable to produce infectious virusfollowing introduction into a cell lacking the complementary geneticinformation enabling encapsidation.

Encapsidation in a cell line harboring transacting elements encoding anecotropic viral envelope (e.g., ψ2) provides ecotropic (limited hostrange) progeny virus. Alternatively, assembly in a cell line containingamphotropic packaging genes (.e.g., PA317, ATCC CRL 9078) providesamphotropic progeny virus. Miller and Buttimore, Mol. Cell. Biol.,6:2895 (1986). Such packaging cell lines provide the necessaryretroviral gag, pol and env proteins in trans. This strategy results inthe production of retroviral particles which are highly infectious formammalian cells, while being incapable of further replication after theyhave integrated into the genome of the target cell. The product of theenv gene is responsible for the binding of the retrovirus to viralreceptors on the surface of the target cell and therefore determines thehost range of the retrovirus. The PA317 cells produce retroviralparticles with an amphotropic envelope protein, which can transducecells of human and other species origin. Other packaging cell linesproduce particles With ecotropic envelope proteins, which are able totransduce only mouse and rat cells.

Numerous retroviral vector constructs have been used successfully toexpress many foreign genes (see, e.g., Coffin (1985). Retroviral vectorswith inserted sequences are generally functional, and few sequences thatare consistently inhibitory for retroviral infection have beenidentified. Functional polyadenylation motifs inhibit retroviralreplication by blocking retroviral RNA synthesis, and there is an uppersize limit of approximately 11 kb of sequence which can be packaged intoretroviral particles; however, the presence of multiple internalpromoters, initially thought to be problematic, was found to be welltolerated in several retroviral constructs. Coffin (1985); and Overellet al., Mol. Cell. Biol., 8:1803 (1983).

Many gene products have been expressed in retroviral vectors. This caneither be achieved by placing the sequences to be expressed under thetranscriptional control of the promoter incorporated in the retroviralLTR, or by placing them under the control of a heterologous promoterinserted between the LTRs. The latter strategy provides a way ofcoexpressing a dominant selectable marker gene in the vector, thusallowing selection of cells which are expressing specific vectorsequences.

In other embodiments of the invention, the recombinant polynucleotidesencoding the costimulatory factor and target antigen polypeptide areintroduced directly into the individual to be treated and/or immunized.In one embodiment the polynucleotides of the invention are administereddirectly to the individual to be treated. In this method it is preferredthat the polynucleotide encode both the costimulatory factor and thetarget antigen polypeptide. In addition, it is preferred that thepolynucleotide be in the form of an expression vector.

The polynucleotides are mixed with suitable excipients, and administeredto the individual by any suitable means known in the art, including, forexample parenteral (including, for example, intravenous,intraperitoneal, intramuscular, and subcutaneous) ingestion,lipofection, and transdermal. Suitable excipients are known in the art,and my be dependent upon the species of the individual to which thepolynucleotides are administered as well as the mode of administration.The amount of polynucleotide to be administered to the individual is anamount sufficient to render immunity to the immunized individual. Thisamount will vary depending upon the individual treated, and will bedetermined by the physician or veterinarian rendering the treatment. Theamount to be administered as well as the time of administration (priorto or post-infection) and the number of doses whether single ormultiple, is determined by routine methods known to those of skill inthe art.

In another embodiment of the invention, the polynucleotides of theinvention are encapsidated in virions, and the individual is treatedwith the encapsidated polynucleotide. The virions used my be any ofthose that are known in the art to be suitable for gene therapyprocedures, several of which are discussed above for the introduction ofthe polynucleotides into a cell ex vivo. The mode of administration isdependent upon the virion used, and my include, for example, thoselisted above for administration of unencapsidated polynucleotides. Thevirions are prepared in a suitable excipient, and administered to theindividual. The amount to be administered as well as the time ofadministration (prior to or post-infection) and the number of doseswhether single or multiple, is determined by the administering physicianor veterinarian, and is obtainable by routine methods known to those ofskill in the art.

The examples presented below are provided as a further guide to thepractitioner of ordinary skill in the art, and are not to be construedas limiting the invention in any way.

EXAMPLE 1 Design of an Expression Vector Encoding a B7 Polypeptide andGSVH c-polypeptide as a Viral Target-Antigen Polypeptide

Chronic ground squirrel hepatitis virus (GSVH) infection of Beecheyground squirrels is a model system for HBV infection in humans. (Cf.Seeger et al., J. Virol., 51:367-375 (1984)). The present exampledescribes a retroviral vector construct for expression of the B7 geneand a gene encoding the c-antigen (cAg) of GSVH.

The vector construct utilizes the Moloney murine leukemia virus (MMLV)based replication-incompetent vector pMV-7 (Ausubel et al. (1989); andKirschmeier et al., DNA, 2:219-225 (1988)). pMV-7 contains a neomycinresistant gene under the control of the HSV thymidine kinase (TK)promoter, thus allowing selection in tissue culture of cells containingthe vector.

The GSHV c-gene including the precore sequence is used as apolynucleotide sequence encoding a target antigen. The GSVH c-gene isisolated from the viral genome (EMBC/Genbank Accession Number K02715) bypolymerase chain reaction (PCR). Marion et al., Proc. Natl. Acad. Sci.USA, 77.:2941-2945 (1980) and Seeger. (1984). The isolated. GSHV c-geneis then introduced into the pMV-7 vector under Control of the MMLV LTR.

Human B7 cDNA (EMBL/Genbank Accession Number M27533) is cloned as cDNAamplified by PCR from the B7 mRNA of the Raji human B cell line usingprimers designed from the published cDNA sequence. Freeman (1989). cDNAencoding the B7-1 gene was introduced into the same vector as the GSVHc-gene under control by the CMV immediate early promoter. Gaballe etal., J. Virol., 62:3334-3340 (1988).

The pMV-7 based vector construct encoding the B7 polypeptide and cAgpolypeptide was transfected into a retroviral packaging cell line,psi-2. Miller et al., Mol. Cell. Biol.., 6:2895-2902 (1986). Thetransfected cells are then selected by growing in a culture mediumcontaining neomycin. Replication-incompetent retrovirus with anamphotropic envelope produced by the cells grown in the neomycin culturewere found in the culture medium.

EXAMPLE 2 Design of an Expression Vector Encoding a B7 polypeptide andGSVH GSHsAg-polypeptide as a Viral Target-Antigen Polypeptide

The construct is prepared as in Example 1, except that the GSVHpolynucleotide encoding ground squirrel hepatitis surface antigen(GSHsAg) including the pre-S1, pre-S2 and S regions is substituted forthat encoding GSVH cAg. The polynucleotide sequence encoding theaforementioned S regions is described in Seeger et al. (1984).

EXAMPLE 3 Use of a Vector Encoding B7 Polypeptide and GSHsAg to ConferImmunity to Uninfected Individuals to GSHV

A therapeutically effective amount of a composition comprised of aninfectious retroviral vector packaged in an amphotropic envelope (10⁷tissue culture infectious units) containing polynucleotide sequencesencoding a B7 polypeptide and a GSHsAg polypeptide is administeredparenterally to an uninfected and GSHV susceptible Beechey groundsquirrel. Seeger et al. (1984). The vector is of the constructiondescribed in Example 2. An "uninfected" GSHV susceptible ground squirrelhas no detectable serum antibody to ground squirrel hepatitis virus coreantigen (anti-GSHcAg) or antibody to ground squirrel hepatitis virussurface antigen (anti-GSHsAg). The parenteral administration of aretroviral vector containing the B7 gene and GSHV gene results in aserum anti-GSHsAg response and a CTL response directed at cellsexpressing GSHsAg.

The dual response, i.e., serum and CTL, confers at least partialimmunity against future infection by GSHV on the uninfected groundsquirrel.

EXAMPLE 4 Use of A Vector Encoding B7 Polypeptide and GSHsAg Polypeptidefor Treatment of Chronic Virus Infection

Two different methods my be used to administer polynucleotides encodinga B7 polypeptide and a viral target antigen to animals chronicallyinfected with GSHV for treatment of an infection.

Delivery of the Vector Encoding the B7 polypeptide and GSHsAgPolypeptide Directly to an Infected Mammal

An infectious retroviral vector packaged in an amphotropic envelope andencoding a B7 polypeptide and a GSHcAg polypeptide are constructed as inExample 1. The vector is administered parenterally in a therapeuticallyeffective amount to a ground squirrel chronically infected with GSHV.

The effect of the expression of polypeptides from the retroviral vectoron the immune response by the ground squirrel, including a CTL responsedirected at cells expressing GSHcAg, is monitored. In addition, theeffect on the chronicity of the disease, including the presence of viralDNA, the presence of GSHsAg and pathogenic effects associated with thedisease is monitored. Diminution in physical symptomology and/or inGSHsAg and/or in GVSH DNA in the treated individual is indicative ofalleviation and/or termination of the chronic disease caused by GSHV.

Treatment of an Infected Mammal with Cells Transfected ex vivo with aVector Encoding B7 Polypeptide and GSH cAg

Fibroblast cells isolated from a skin biopsy of the infected individualare grown in culture, and are transfected with a retroviral vectorencoding a B7 polypeptide and a GSHcAg polypeptide. The vector isconstructed as described in Example 1. Infected (transduced) cells arethen selected by growing cells in a culture medium containing neomycin.The cells expressing GSHcAg are identified by using fluorescent staining(IFA) with anti-HBc and B7 protein expression using ELISA with anti-B7monoclonal antibody (mAb). A therapeutically effective amount of theautologous cells expressing GSHcAg and B7 are infused intravenously intothe GSHV infected ground squirrel.

The effect of the expression of polypeptides from the retroviral vectorsin the-implanted cells on the immune response by the ground squirrel,including a CTL response directed at cells expressing GSHcAg, ismonitored. In addition, the effect on the chronicity of the disease,including the presence of viral DNA, the presence of GSHsAg andpathogenic effects associated with the disease is monitored. Diminutionin physical symptomology and/or in GSHsAg and/or in GVSH DNA in thetreated individual is indicative of alleviation and/or termination ofthe chronic disease caused by GSHV.

EXAMPLE 5

Construction of retroviral vectors to express B7 and HBS

FIG. 1 depicts recombinant retroviral vectors for the demonstration ofthe enhancement of the immune response to hepatitis B virus surfaceantigen (HBS) by co-expression with B7-1 in cells in vivo. Retroviralvector pMV-7 DNA was used to construct recombinant vectors 907 with theHBS coding sequence inserted at the polylinker of pMV-7 ; andrecombinant vector 1016 with the HBS coding sequence, a cap independenttranslation element (CITE) of encephalomycoarditis virus (EMC) and thecoding sequence for murine B7-1 all in the same reading frame insertedat the polylinker of pMV-7. Each recombinant vector was used totransfect by the calcium phosphate DNA method a murine Balbc packagingcell line and cells containing the respective vectors were selected bygrowth in cell culture medium containing neomycin. The packaging cellpopulation neomycin selected to contain recombinant vector 907 and thoseselected for recombinant vector 1016 released virus that could beassayed by the ability to confer neomycin resistance to Balbc 3T3 cells.Neomycin resistant Balbc 3T3 cells infected with recombinant virus 907were shown to release HBS by ELISA. Neomycin resistant Balbc 3T3 cellsinfected with recombinant virus 1016 were shown to express HBS as aboveand the B7-1 protein by FACS analysis using monoclonal antibody to theB7-1 protein. Cells (2×107) of each respective type and nontransfectedBalbc 3T3 cells were each inoculated into seperate groups of 10 Balbcmice by the intrapertioneal route for study of the immune response toHBS. Anti-HBS measured by ELISA, proliferation of spleen cells exposedto HBS in vitro and cytotoxic activity of spleen cells by chromium51release from Balbc 3T3 cells expressing HBS are assayed in 5 mice ofeach group at 2 weeks and 5 mice of each group at 4 weeks postinoculation. In FIG. 1, LTR represents the long terminal repeats of theretroviral vector, TK represents the thymidine kinase promoter of herpessimplex virus, Neo represents a gene conferring neomycin resistance.

Although the foregoing invention has been described in some detail bywayof illustration and example for purposes of clarity and understanding,it will be apparent to those skilled in the art that certain changes andmodifications may be practiced. Therefore, the description and examplesshould not be construed as limiting the scope of the invention which isdelineated by the appended claims.

What is claimed:
 1. A method of enhancing an antigen-specific T cellresponse in an individual to a target antigen comprising administeringat least one polynucleotide sequence encoding at least one targetantigen or a portion of the target antigen and at least onepolynucleotide sequence encoding at least one B7 costimulatory moleculethat interacts with T cell surface molecules for T cell activation, saidpolynucleotide sequences expressed in an amount effective to enhance a Tcell response to eliminate cells bearing said target antigen or portionthereof, when said polynucleotide sequence encoding the target antigenand said polynucleotide sequence encoding the B7 costimulatory moleculeare expressed in said individual.
 2. The method according to claim 1,wherein said B7 costimulatory molecule is selected from the groupconsisting of B7-1, B7-2 and B7-3 molecules.
 3. The method of claim 1wherein said target antigen is an antigen of an infectious agent.
 4. Themethod of claim 3 wherein said infectious agent is selected from thegroup consisting of viruses, protozoans, fungi, bacteria, parasites andworms.
 5. The method of claim 1 wherein said polynucleotide sequenceencoding the target antigen or a portion of the target antigen isoperably linked to a transcriptional control region in a vector.
 6. Themethod of claim 1 wherein said polynucleotide sequence encoding the B7costimulatory molecule is operably linked to a transcriptional controlregion in a vector.
 7. The method of claim 1 wherein said polynucleotidesequence encoding the target antigen or a portion of the target antigenis operably linked to a transcriptional control region in a vector andsaid polynucleotide sequence encoding the B7 costimulatory molecule isoperably linked to a transcriptional control region in said vector. 8.The method according to claim 5, 6 or 7 wherein said vector is a vitalvector.
 9. The method of claim 5, 6 or 7 wherein said vector is apolynucleotide vector.
 10. A vector comprising at least onepolynucleotide sequence encoding at least one target antigen or encodinga portion of said target antigen and at least one polynucleotidesequence encoding at least one B7 costimulatory molecule that interactswith T cell surface molecules for T cell activation when said B7costimulatory molecule and said antigen are both expressed in anindividual, wherein said polynucleotide sequences are expressed in anamount effective to enhance the T cell response to eliminate cellsbearing said target antigen or portion thereof, and wherein saidpolynucleotide sequence encoding the target antigen is operably linkedto a transcriptional control region and said polynucleotide sequenceencoding the B7 costimulatory molecule is operably linked to atranscriptional control region for expression in said individual. 11.The vector of claim 10, wherein said B7 costimulatory molecule isselected from the group consisting of B7-1, B7-2 and B7-3 molecules. 12.The vector of claim 10 wherein said vector is a viral vector.
 13. Thevector of claim 10 wherein said vector is a polynucleotide vector. 14.The vector of claim 10 wherein said target antigen is an antigen of aninfectious agent.
 15. The vector of claim 14 wherein said infectiousagent is selected from the group consisting of viruses, protozoans,fungi, bacteria, parasites and worms.